Antibodies to granulocyte-macrophage colony-stimulating factor

ABSTRACT

The current invention relates to high-affinity antibodies to Granulocyte-Macrophage Colony-Stimulating Factor that have reduced immunogenicity when administered to a human to treat diseases and method of using such antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional application No.61/048,522, filed Apr. 28, 2008, which application is hereinincorporated by reference.

BACKGROUND OF THE INVENTION

“Granulocyte Macrophage-Colony Stimulating Factor” (GM-CSF) is a smallglycoprotein that is produced in response to a number of inflammatorymediators by cells present in the hemopoietic environment and atperipheral sites of inflammation. GM-CSF is able to stimulate theproduction of neutrophilic granulocytes, macrophages, and mixedgranulocyte-macrophage colonies from bone marrow cells and can stimulatethe formation of eosinophil colonies from fetal liver progenitor cells.GM-CSF can also stimulate some functional activities in maturegranulocytes and macrophages and inhibits apoptosis of granulocytes andmacrophages.

GM-CSF has been proposed to play a role in the pathogenesis of a numberof diseases. There is therefore a need for additional therapies thattarget GM-CSF. The current invention provides improved anti-GM-CSFantibodies, e.g., for the treatment of diseases in which GM-CSF is partof the pathogenic mechanism.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides anti-GM-CSF antibodies, e.g.,humaneered antibodies, and methods of using such antibodies for thetreatment of diseases for which it is desirable to inhibit GM-CSFreceptor signaling. Thus, in some embodiments, the invention provides ananti-GM-CSF antibody, comprising a heavy chain variable region thatcomprises a CDR3 binding specificity determinant RQRFPY (SEQ ID NO:12)or RDRFPY (SEQ ID NO:13), a J segment, and a V-segment, wherein theJ-segment comprises at least 95% identity to human JH4 (YFDYWGQGTLVTVSS;SEQ ID NO:14) and the V-segment comprises at least 90% identity to ahuman germ line VH1 1-02 or VH1 1-03 sequence; or a heavy chain variableregion that comprises a CDR3 binding specificity determinant comprisingRQRFPY (SEQ ID NO:12). In some embodiments, the J segment comprisesYFDYWGQGTLVTVSS (SEQ ID NO:14). In some embodiments, the CDR3 comprisesRQRFPYYFDY (SEQ ID NO:15) or RDRFPYYFDY (SEQ ID NO:16). In someembodiments, the heavy chain variable region CDR1 or CDR2 can be a humangermline VH1 sequence; or both the CDR1 and CDR2 can be human germlineVH1. In some embodiments, the antibody comprises a heavy chain variableregion CDR1 or CDR2, or both CDR1 and CDR2, as shown in a V_(H) regionset forth in FIG. 1. In some embodiments, an antibody of the inventionas a V-segment that has a V_(H) V-segment sequence shown in FIG. 1. Insome embodiments, an antibody of the invention comprises a V_(H) thathas the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 set forth in FIG. 1.

The invention also provides an anti-GM-CSF antibody, e.g., that has aheavy chain variable region as described in the paragraph above, wherethe antibody comprises a light chain variable region that comprises aCDR3 binding specificity determinant FNK or FNR. In some embodiments,such an antibody comprises a human germline JK4 region. In someembodiments, the antibody V_(L) region CDR3 comprises QQFN(K/R)SPLT (SEQID NO:17), e.g., QQFNKSPLT (SEQ ID NO:18). In some embodiments, thelight chain variable region comprises a CDR1, or a CDR2, or both a CDR1and CDR2, of a V_(L) region shown in FIG. 1. In some embodiments, theV_(L) region comprises a V segment that has at least 95% identity to theVKIIIA27 V-segment sequence as shown in FIG. 1. In some embodiments, theV_(L) region has the sequence of VK#1, VK#2, VK#3, or VK#4 set forth inFIG. 1.

In some embodiments, an antibody of the invention, e.g., that has aV_(H) region sequence selected from the V_(H) region sequences in FIG. 1and a V_(L) region selected from the V_(L) region sequences in FIG. 1,have a monovalent affinity better than about 10 nM, and often better(less) than about 500 pM or better than about 50 pM as determined bysurface plasmon resonance analysis performed at 37° C. Thus, in someembodiments, the antibodies of the invention have an affinity (asmeasured using surface plasmon resonance), of less than 50 pM, typicallyless than about 25 pM, or even less than 10 pM. In some embodiments, ananti-GM-CSF of the invention has a slow dissociation rate with adissociation rate constant (kd) determined by surface plasmon resonanceanalysis at 37° C. for the monovalent interaction with GM-CSF less thanapproximately 10⁻⁴ s⁻¹, preferably less than 5×10⁻⁵ s⁻¹ and mostpreferably less than 10⁻⁵ s⁻¹. In some embodiments, an antibody of theinvention has a dissociation rate that is at least 2 to 3-fold slowerthan a reference chimeric c19/2 monoclonal antibody assayed under thesame conditions, but has a potency that is at least 6-10 times greaterthan that of the reference antibody in neutralizing GM-CSF activity in acell-based assay that measures GM-CSF activity.

In some embodiments, an antibody of the invention having a heavy chainvariable region and/or a light chain variable region as described hereinis an IgG. In some embodiments, the heavy chain has a constant regionthat has the amino acid sequence of SEQ ID NO:11. In some embodiments,the light chain constant region is a kappa light chain having a sequenceset forth in SEQ ID NO:10. In some embodiments, an antibody of theinvention has a heavy chain constant region as set forth in SEQ IDNO:11, a light chain kappa constant region having a sequence as setforth in SEQ ID NO:10 and a heavy chain and light chain variable regionselected from the heavy and light chain variable regions shown in FIG.1, e.g., the antibody heavy and light chain variable regions compriseone of the following combinations from the sequences set forth in FIG.1: a) VH#2, VK#3; b) VH#1, VK#3; c) VH#3, VK#1; d) VH#4, VK#3; e) VH#4,VK#4; f) VH#4, VK#2; g) VH#5, VK#1; h) VH#5, VK#2; i) VH#3, VK#4; or j)VH#3, VL#3). One of skill will recognize that antibodies of theinvention can be selected from any of the combinations of V_(H) andV_(L) regions set forth in FIG. 1 in conjunction with the teachingsherein.

In some embodiments, the V_(H) region sequence or V_(L) region sequence,or both the V_(H) and V_(L) region amino acid sequences, comprise amethionine at the N-terminus.

In additional aspects, the invention provides a method of treating apatient that has a disease in which it is desirable to inhibit GM-CSF,the method comprising administering an antibody of any one of thepreceding claims to the patient in a therapeutically effective amount.In some embodiments, the patient has osteopenia, rheumatoid arthritis,asthma, multiple sclerosis, psoriasis, chronic obstructive pulmonarydisease, idiopathic thrombocytopenia purpura, Alzheimer's disease, heartfailure, cardiac damage due to an ischemic event, or diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides exemplary V_(H) (SEQ ID NOS:1-5) and V_(L) (SEQ IDNOS:6-9) sequences of anti-GM-CSF antibodies. VH1 1-02=SEQ ID NO:19; VH11-03=SEQ ID NO:20; VKIII A27=SEQ ID NO:21.

FIG. 2. Binding of GM-CSF to Ab1 (A) or Ab2 (B) determined by surfaceplasmon resonance analysis at 37° C. (Biacore 3000). Ab1 and Ab2 werecaptured on anti Fab polyclonal antibodies immobilized on the Biacorechip. Different concentrations of GM-CSF were injected over the surfaceas indicated. Global fit analysis was carried out assuming a 1:1interaction using Scrubber2 software.

FIG. 3. Binding of Ab1 and Ab2 to glycosylated and non-glycosylatedGM-CSF. Binding to glycosylated GM-CSF expressed from human 293 cells ornon-glycosylated GM-CSF expressed in E. coli was determined by ELISA.Representative results from a single experiment are shown (exp 1).Two-fold dilutions of Ab1 and Ab2 starting from 1500 ng/ml were appliedto GM-CSF coated wells. Each point represents mean±standard error fortriplicate determinations. Sigmoidal curve fit was performed using Prism5.0 Software (Graphpad).

FIG. 4. Competition ELISA demonstrating binding of Ab1 and Ab2 to ashared epitope. ELISA plates coated with 50 ng/well of recombinantGM-CSF were incubated with various concentrations of antibody (Ab2, Ab1or isotype control antibody) together with 50 nM biotinylated Ab2.Biotinylated antibody binding was assayed using neutravidin-HRPconjugate. Competition for binding to GM-CSF was for 1 hr (A) or for 18hrs (B). Each point represents mean±standard error for triplicatedeterminations. Sigmoidal curve fit was performed using Prism 5.0Software (Graphpad).

FIG. 5. Inhibition of GM-CSF-induced IL-8 expression. Various amounts ofeach antibody were incubated with 0.5 ng/ml GM-CSF and incubated withU937 cells for 16 hrs. IL-8 secreted into the culture supernatant wasdetermined by ELISA

DETAILED DESCRIPTION OF THE INVENTION

As used herein, an “antibody” refers to a protein functionally definedas a binding protein and structurally defined as comprising an aminoacid sequence that is recognized by one of skill as being derived fromthe framework region of an immunoglobulin-encoding gene of an animalthat produces antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains, respectively.

The term “antibody” as used herein also includes antibody fragments thatretain binding specificity. For example, there are a number of wellcharacterized antibody fragments. Thus, for example, pepsin digests anantibody C-terminal to the disulfide linkages in the hinge region toproduce F(ab′)2, a dimer of Fab which itself is a light chain joined toVH-CH1 by a disulfide bond. The F(ab′)2 may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)2 dimer into a Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that fragments can be synthesizedde novo by utilizing recombinant DNA methodology or chemically. Thus,the term “antibody”, as used here includes antibody fragments eitherproduced by the modification of whole antibodies or synthesized usingrecombinant DNA methodologies.

Antibodies as used here also include various V_(H)-V_(L) pair formats,including single chain antibodies (antibodies that exist as a singlepolypeptide chain), e.g., single chain Fv antibodies (sFv or scFv), inwhich a variable heavy and a variable light region are joined together(directly or through a peptide linker) to form a continuous polypeptide.The single chain Fv antibody is a covalently linked V_(H)-V_(L) that maybe expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker(e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988).While the V_(H) and V_(L) are connected to each as a single polypeptidechain, the V_(H) and V_(L) domains associate non-covalently. An antibodycan also be in another fragment form, such as a disulfide-stabilized Fv(dsFv). Other fragments can also be generated, e.g., using recombinanttechniques, as soluble proteins or as fragments obtained from displaymethods. Antibodies can also include diantibodies and miniantibodies.

Antibodies of the invention also include heavy chain dimers, such asantibodies from camelids. Since the V_(H) region of a heavy chain dimerIgG in a camelid does not have to make hydrophobic interactions with alight chain, the region in the heavy chain that normally contacts alight chain is changed to hydrophilic amino acid residues in a camelid.V_(H) domains of heavy-chain dimer IgGs are called VHH domains.Antibodies for use in the current invention additionally include singledomain antibodies (dAbs) and nanobodies (see, e.g., Cortez-Retamozo, etal., Cancer Res. 64:2853-2857, 2004).

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework 3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation. A “V-segment”as used herein refers to the region of the V-region (heavy or lightchain) that is encoded by a V gene. The V-segment of the heavy chainvariable region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes ofthis invention, the V-segment of the light chain variable region isdefined as extending though FR3 up to CDR3.

As used herein, the term “J-segment” refers to a subsequence of thevariable region encoded comprising a C-terminal portion of a CDR3 andthe FR4. An endogenous J-segment is encoded by an immunoglobulin J-gene.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, for example, a V_(H) CDR3 is located in the variabledomain of the heavy chain of the antibody in which it is found, whereasa V_(L) CDR1 is the CDR1 from the variable domain of the light chain ofthe antibody in which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structuresfor the hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol. 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1): 207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl. Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

The term “binding specificity determinant” or “BSD” as used in thecontext of the current invention refers to the minimum contiguous ornon-contiguous amino acid sequence within a CDR region necessary fordetermining the binding specificity of an antibody. In the currentinvention, the minimum binding specificity determinants reside within aportion or the full-length of the CDR3 sequences of the heavy and lightchains of the antibody.

As used herein, “anti-GM-CSF antibody” or “GM-CSF antibody” are usedinterchangeably to refer to an antibody that binds to GM-CSF andinhibits GM-CSF receptor activity. Such antibodies may be identifiedusing any number of art-recognized assays that assess GM-CSF bindingand/or function. For example, binding assays such as ELISA assays thatmeasure the inhibition of GM-CSF binding to the alpha receptor subunitmay be used. Cell-based assays for GM-CSF receptor signaling, such asassays which determine the rate of proliferation of a GM-CSF-dependentcell line in response to a limiting amount of GM-CSF, are alsoconveniently employed, as are assays that measure amounts of cytokineproduction, e.g., IL-8 production, in response to GM-CSF exposure.

As used herein, “neutralizing antibody” refers to an antibody that bindsto GM-CSF and inhibits signaling by the GM-CSF receptor, or inhibitsbinding of GM-CSF to its receptor.

As used herein, “Granulocyte Macrophage-Colony Stimulating Factor”(GM-CSF) refers to a small naturally occurring glycoprotein withinternal disulfide bonds having a molecular weight of approximately 23kDa. In humans, it is encoded by a gene located within the cytokinecluster on human chromosome 5. The sequence of the human gene andprotein are known. The protein has an N-terminal signal sequence, and aC-terminal receptor binding domain (Rasko and Gough In: The CytokineHandbook, A. Thomson, et al, Academic Press, New York (1994) pages349-369). Its three-dimensional structure is similar to that of theinterleukins, although the amino acid sequences are not similar. GM-CSFis produced in response to a number of inflammatory mediators present inthe hemopoietic environment and at peripheral sites of inflammation.GM-CSF is able to stimulate the production of neutrophilic granulocytes,macrophages, and mixed granulocyte-macrophage colonies from bone marrowcells and can stimulate the formation of eosinophil colonies from fetalliver progenitor cells. GM-CSF can also stimulate some functionalactivities in mature granulocytes and macrophages and inhibits apoptosisof granulocytes and macrophages.

The term “equilibrium dissociation constant” or “affinity” abbreviated(K_(D)), refers to the dissociation rate constant (k_(d), time⁻¹)divided by the association rate constant (k_(a), time⁻¹M⁻¹). Equilibriumdissociation constants can be measured using any known method in theart. The antibodies of the present invention are high affinityantibodies. Such antibodies have a monovalent affinity better (less)than about 10 nM, and often better than about 500 pM or better thanabout 50 pM as determined by surface plasmon resonance analysisperformed at 37° C. Thus, in some embodiments, the antibodies of theinvention have an affinity (as measured using surface plasmonresonance), of less than 50 pM, typically less than about 25 pM, or evenless than 10 pM.

In some embodiments, an anti-GM-CSF of the invention has a slowdissociation rate with a dissociation rate constant (kd) determined bysurface plasmon resonance analysis at 37° C. for the monovalentinteraction with GM-CSF less than approximately 10⁻⁴ s⁻¹, preferablyless than 5×10⁻⁵ s⁻¹ and most preferably less than 10⁻⁵ S⁻¹.

As used herein, “humanized antibody” refers to an immunoglobulinmolecule in CDRs from a donor antibody are grafted onto human frameworksequences. Humanized antibodies may also comprise residues of donororigin in the framework sequences. The humanized antibody can alsocomprise at least a portion of a human immunoglobulin constant region.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. Humanization can be performed using methods known in the art(e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988);Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No.4,816,567), including techniques such as “superhumanizing” antibodies(Tan et al., J. Immunol. 169: 1119, 2002) and “resurfacing” (e.g.,Staelens et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.Natl. Acad. Sci. USA 91: 969, 1994).

A “humaneered” antibody in the context of this invention refers to anengineered human antibody having a binding specificity of a referenceantibody. A “humaneered” antibody for use in this invention has animmunoglobulin molecule that contains minimal sequence derived from adonor immunoglobulin. Typically, an antibody is “humaneered” by joininga DNA sequence encoding a binding specificity determinant (BSD) from theCDR3 region of the heavy chain of the reference antibody to human V_(H)segment sequence and a light chain CDR3 BSD from the reference antibodyto a human V_(L) segment sequence. A “BSD” refers to a CDR3-FR4 region,or a portion of this region that mediates binding specificity. A bindingspecificity determinant therefore can be a CDR3-FR4, a CDR3, a minimalessential binding specificity determinant of a CDR3 (which refers to anyregion smaller than the CDR3 that confers binding specificity whenpresent in the V region of an antibody), the D segment (with regard to aheavy chain region), or other regions of CDR3-FR4 that confer thebinding specificity of a reference antibody. Methods for humaneering areprovided in US patent application publication no. 20050255552 and USpatent application publication no. 20060134098.

A “human” antibody as used herein encompasses humanized and humaneeredantibodies, as well as human monoclonal antibodies that are obtainedusing known techniques.

The term “hybrid” when used with reference to portions of a nucleic acidor protein, indicates that the nucleic acid or protein comprises two ormore subsequences that are not normally found in the same relationshipto each other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid.Similarly, a hybrid protein refers to two or more subsequences that arenot normally found in the same relationship to each other in nature.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction where the antibodybinds to the protein of interest. In the context of this invention, theantibody binds to the antigen of interest, e.g., GM-CSF, with anaffinity that is at least 100-fold better than its affinity for otherantigens.

The terms “identical” or percent “identity,” in the context of two ormore polypeptide (or nucleic acid) sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues (or nucleotides) that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site). Such sequences are then said to be “substantiallyidentical.” “Substantially identical” sequences also includes sequencesthat have deletions and/or additions, as well as those that havesubstitutions, as well as naturally occurring, e.g., polymorphic orallelic variants, and man-made variants. As described below, thepreferred algorithms can account for gaps and the like. Preferably,protein sequence identity exists over a region that is at least about 25amino acids in length, or more preferably over a region that is 50-100amino acids=in length, or over the length of a protein.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990). BLAST and BLAST 2.0 are used, with the parameters describedherein, to determine percent sequence identity for the nucleic acids andproteins of the invention. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. The term“purified” in some embodiments denotes that a protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the protein is at least 85% pure, more preferably at least 95%pure, and most preferably at least 99% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables and substitution matrices such asBLOSUM providing functionally similar amino acids are well known in theart. Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. Typical conservative substitutions for one anotherinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

I. Introduction

The invention relates to antibodies that bind with high affinity toGM-CSF and are antagonists of GM-CSF. The antibodies comprise variableregions with a high degree of identity to human germ-line V_(H) andV_(L) sequences. In preferred embodiments, the BSD sequence in CDRH3 ofan antibody of the invention comprises the amino acid sequence RQRFPY(SEQ ID NO:12) or RDRFPY (SEQ ID NO:13). The BSD in CDRL3 comprises FNKor FNR.

Complete V-regions are generated in which the BSD forms part of the CDR3and additional sequences are used to complete the CDR3 and add a FR4sequence. Typically, the portion of the CDR3 excluding the BSD and thecomplete FR4 are comprised of human germ-line sequences. In someembodiments, the CDR3-FR4 sequence excluding the BSD differs from humangerm-line sequences by not more than 2 amino acids on each chain. Insome embodiments, the J-segment comprises a human germline J-segment.Human germline sequences can be determined, for example, through thepublicly available international ImMunoGeneTics database (IMGT) andV-base (on the worldwide web at vbase.mrc-cpe.cam.ac.uk).

The human germline V-segment repertoire consists of 51 heavy chainV-regions, 40κ light chain V-segments, and 31λ light chain V-segments,making a total of 3,621 germline V-region pairs. In addition, there arestable allelic variants for most of these V-segments, but thecontribution of these variants to the structural diversity of thegermline repertoire is limited. The sequences of all human germ-lineV-segment genes are known and can be accessed in the V-base database,provided by the MRC Centre for Protein Engineering, Cambridge, UnitedKingdom (see, also Chothia et al., 1992, J Mol Biol 227:776-798;Tomlinson et al., 1995, EMBO J 14:4628-4638; and Williams et al., 1996,J Mol Biol 264:220-232).

Antibodies or antibodies fragments as described herein can be expressedin prokaryotic or eukaryotic microbial systems or in the cells of highereukaryotes such as mammalian cells.

An antibody that is employed in the invention can be in any format. Forexample, in some embodiments, the antibody can be a complete antibodyincluding a constant region, e.g., a human constant region, or can be afragment or derivative of a complete antibody, e.g., a Fab, Fab′,F(ab′)₂, scFv, Fv, or a single domain antibody, such as a nanobody or acamelid antibody.

II. Heavy Chains

A heavy chain of an anti-GM-CSF antibody of the invention comprises aheavy-chain V-region that comprises the following elements:

1) human heavy-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRH3 region comprising the amino acid sequence R(Q/D)RFPY (SEQ IDNO:22)

3) a FR4 contributed by a human germ-line J-gene segment.

Examples of V-segment sequences that support binding to GM-CSF incombination with a CDR3-FR4 segment described above together with acomplementary V_(L) region are shown in FIG. 1. The V-segments can be,e.g., from the human VH1 subclass. In some embodiments, the V-segment isa human V_(H)1 sub-class segment that has a high degree of amino-acidsequence identity, e.g., at least 80%, 85%, or 90% or greater identity,to the germ-line segment VH1 1-02 or VH1 1-03. In some embodiments, theV-segment differs by not more than 15 residues from VH1 1-02 or VH1 1-03and preferably not more than 7 residues.

The FR4 sequence of the antibodies of the invention is provided by ahuman JH1, JH3, JH4, JH5 or JH6 gene germline segment, or a sequencethat has a high degree of amino-acid sequence identity to a humangermline JH segment. In some embodiments, the J segment is a humangermline JH4 sequence.

The CDRH3 also comprises sequences that are derived from a humanJ-segment. Typically, the CDRH3-FR4 sequence excluding the BSD differsby not more than 2 amino acids from a human germ-line J-segment. Intypical embodiments, the J-segment sequences in CDRH3 are from the sameJ-segment used for the FR4 sequences. Thus, in some embodiments, theCDRH3-FR4 region comprises the BSD and a complete human JH4 germ-linegene segment. An exemplary combination of CDRH3 and FR4 sequences isshown below (SEQ ID NO:23), in which the BSD is in bold and humangerm-line J-segment JH4 residues are underlined:

        CDR3     R (Q/D) RFPY YFDYWGQGTLVTVSS

In some embodiments, an antibody of the invention comprises a V-segmentthat has at least 90% identity, or at least 91%, 92% 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the germ-line segment VH 1-02 orVH1-03; or to one of the V-segments of the V_(H) regions shown in FIG.1, such as a V-segment portion of VH#1, VH#2, VH#3, VH#4, or VH#5.

In some embodiments, the V-segment of the V_(H) region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 that has the sequence GYYMH (SEQ ID NO:24) or NYYIH (SEQ IDNO:25); or a CDR2 that has the sequence WINPNSGGTNYAQKFQG (SEQ ID NO:26)or WINAGNGNTKYSQKFQG (SEQ ID NO:27).

In particular embodiments, an antibody has both a CDR1 and a CDR2 fromone of the V_(H) region V-segments shown in FIG. 1 and a CDR3 thatcomprises R(Q/D)RFPY (SEO ID NO:22), e.g., RDRFPYYFDY (SEQ ID NO:16) orRQRFPYYFDY (SEQ ID NO:15). Thus, in some embodiments, an anti-GM-CSFantibody of the invention, may for example, have a CDR3-FR4 that has thesequence R(Q/D)RFPYYFDYWGQGTLVTVSS (SEQ ID NO:23) and a CDR1 and/or CDR2as shown in FIG. 1.

In some embodiments, a V_(H) region of an antibody of the invention hasa CDR3 that has a binding specificity determinant R(Q/D)RFPY (SEQ IDNO:22), a CDR2 from a human germline VH1 segment or a CDR1 from a humangermline VH1. In some embodiments, both the CDR1 and CDR2 are from humangermline VH1 segments.

III. Light Chains

A light chain of an anti-GM-CSF antibody of the invention comprises atlight-chain V-region that comprises the following elements:

1) human light-chain V-segment sequences comprisingFR1-CDR1-FR2-CDR2-FR3

2) a CDRL3 region comprising the sequence FNK or FNR, e.g., QQFNRSPLT(SEQ ID NO:28) or QQFNKSPLT (SEQ ID NO:18).

3) a FR4 contributed by a human germ-line J-gene segment.

The V_(L) region comprises either a Vlambda or a Vkappa V-segment. Anexample of a Vkappa sequence that supports binding in combination with acomplementary V_(H)-region is provided in FIG. 1.

The V_(L) region CDR3 sequence comprises a J-segment derived sequence.In typical embodiments, the J-segment sequences in CDRL3 are from thesame J-segment used for FR4. Thus, the sequence in some embodiments maydiffer by not more than 2 amino acids from human kappa germ-lineV-segment and J-segment sequences. In some embodiments, the CDRL3-FR4region comprises the BSD and the complete human JK4 germline genesegment. Exemplary CDRL3-FR4 combinations for kappa chains are shownbelow in which the minimal essential binding specificity determinant isshown in bold and JK4 sequences are underlined:

  CDR3   QQFNRSPLTFGGGTKVEIK QQFNKSPLTFGGGTKVEIK

The Vkappa segments are typically of the VKIII sub-class. In someembodiments, the segments have at least 80% sequence identity to a humangermline VKIII subclass, e.g., at least 80% identity to the humangerm-line VKIIIA27 sequence. In some embodiments, the Vkappa segment maydiffer by not more than 18 residues from VKIIIA27. In other embodiments,the V_(L) region V-segment of an antibody of the invention has at least85% identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identity to the human kappa V-segment sequence of a V_(L)region shown in FIG. 1, for example, the V-segment sequence of VK#1,VK#2, VK#3, or VK#4.

In some embodiments, the V-segment of the V_(L) region has a CDR1 and/orCDR2 as shown in FIG. 1. For example, an antibody of the invention mayhave a CDR1 sequence of RASQSVGTNVA (SEQ ID NO:31) or RASQSIGSNLA (SEQID NO:32); or a CDR2 sequence STSSRAT (SEQ ID NO:33).

In particular embodiments, an anti-GM-CSF antibody of the invention mayhave a CDR1 and a CDR2 in a combination as shown in one of theV-segments of the V_(L) regions set forth in FIG. 1 and a CDR3 sequencethat comprises FNK or FNR, e.g., the CDR3 may be QQFNKSPLT (SEQ IDNO:18) or QQFNRSPLT (SEQ ID NO:28). In some embodiments, such a GM-CSFantibody may comprise an FR4 region that is FGGGTKVEIK (SEQ ID NO:34).Thus, an anti-GM-CSF antibody of the invention, can comprise, e.g., boththe CDR1 and CDR2 from one of the V_(L) regions shown in FIG. 1 and aCDR3-FR4 region that is FGGGTKVEIK (SEQ ID NO:34).

IV. Preparation of GM-CSF Antibodies

An antibody of the invention may comprise any of the V_(H) regions VH#1,VH#2, VH#3, VH#4, or VH#5 as shown in FIG. 1. In some embodiment, anantibody of the invention may comprise any of the V_(L) regions VK#1,VK#2, VK#3, or VK#4 as shown in FIG. 1. In some embodiments, theantibody has a V_(H) region VH#1, VH#2, VH#3, VH#4, or VH#5 as shown inFIG. 1; and a V_(L) region VK#1, VK#2, VK#3, or VK#4 as shown in FIG. 1.

An antibody may be tested to confirm that the antibody retains theactivity of antagonizing GM-CSF activity. The antagonist activity can bedetermined using any number of endpoints, including proliferationassays. Anti-GM-CSF antibodies may be evaluated using any number ofassays that assess GM-CSF function. For example, cell-based assays forGM-CSF receptor signaling, such as assays which determine the rate ofproliferation of a GM-CSF-dependent cell line in response to a limitingamount of GM-CSF, are conveniently used. The human TF-1 cell line issuitable for use in such an assay. See, Krinner et al., (2007) Mol.Immunol. An antibody that is administered to treat a disease for whichit is desirable to inhibit GM-CSF preferably retains at least about 50%,or at least about 75%, 80%, 90%, 95%, or 100%, of the antagonistactivity of the antibody chimeric c19/2, e.g., WO03/068920, which hasthe variable regions of the mouse monoclonal antibody LMM102 and theCDRs, as defined by Kabat:

CDRH1 DYNIH (SEQ ID NO: 35) CDRH2 YIAPYSGGTGYNQEFKN (SEQ ID NO: 36)CDRH3 RDRFPYYFDY (SEQ ID NO: 16) CDRL1 KASQNVGSNVA (SEQ ID NO: 37) CDRL2SASYRSG (SEQ ID NO: 38) CDRL3 QQFNRSPLT (SEQ ID NO: 28).

A high-affinity antibody may be identified using well known assays todetermine binding activity and affinity. Such techniques include ELISAassays as well as binding determinations that employ surface plasmonresonance or interferometry. For example, affinities can be determinedby biolayer interferometry using a ForteBio (Mountain View, Calif.)Octet biosensor. An antibody of the invention typically binds withsimilar affinity to both glycosylated and non-gycosylated from ofGM-CSF.

Antibodies of the invention compete with c19/2 for binding to GM-CSF.The ability of an antibody described herein to block or compete withc19/2 for binding to GM-CSF indicates that the antibody binds to thesame epitope c19/2 or to an epitope that is close to, e.g., overlapping,with the epitope that is bound by c19/2. In other embodiments anantibody described herein, e.g., an antibody comprising a V_(H) andV_(L) region combination as shown in the table provided in FIG. 1, canbe used as a reference antibody for assessing whether another antibodycompetes for binding to GM-CSF. A test antibody is considered tocompetitively inhibit binding of a reference antibody, if binding of thereference antibody to the antigen is reduced by at least 30%, usually atleast about 40%, 50%, 60% or 75%, and often by at least about 90%, inthe presence of the test antibody. Many assays can be employed to assessbinding, including ELISA, as well as other assays, such as immunoblots.

Methods for the isolation of antibodies with V-region sequences close tohuman germ-line sequences have previously been described (US patentapplication publication nos. 20050255552 and 20060134098). Antibodylibraries may be expressed in a suitable host cell including mammaliancells, yeast cells or prokaryotic cells. For expression in some cellsystems, a signal peptide can be introduced at the N-terminus to directsecretion to the extracellular medium. Antibodies may be secreted frombacterial cells such as E. coli with or without a signal peptide.Methods for signal-less secretion of antibody fragments from E. coli aredescribed in US patent application 20070020685.

To generate a GM-CSF-binding antibody, one of the V_(H)-regions of theinvention, e.g., shown in FIG. 1, is combined with one of theV_(L)-regions of the invention, e.g., shown in FIG. 1, and expressed inany of a number of formats in a suitable expression system. Thus theantibody may be expressed as a scFv, Fab, Fab′ (containing animmunoglobulin hinge sequence), F(ab′)₂, (formed by di-sulfide bondformation between the hinge sequences of two Fab′ molecules), wholeimmunoglobulin or truncated immunoglobulin or as a fusion protein in aprokaryotic or eukaryotic host cell, either inside the host cell or bysecretion. A methionine residue may optionally be present at theN-terminus, for example, in polypeptides produced in signal-lessexpression systems. Each of the V_(H)-regions described herein may bepaired with each of the V_(L) regions to generate an anti-GM-CSFantibody. Exemplary combinations of heavy and light chains are shown inthe table in FIG. 1.

The antibodies of the invention inhibit GM-CSF receptor activation,e.g., by inhibiting GM-CSF binding to the receptor, and exhibit highaffinity binding to GM-CSF, e.g., 500 pM. In some embodiments, theantibody has a dissociation constant of about 10⁻⁴ per sec or less. Notto be bound by theory, an antibody with a slower dissociation constantprovides improved therapeutic benefit. For example, an antibody of theinvention that has a three-fold slower off-rate than c19/2, produced a10-fold more potent GM-CSF neutralizing activity, e.g., in a cell-basedassay such as IL-8 production (see, e.g., Example 2).

Antibodies may be produced using any number of expression systems,including both prokaryotic and eukaryotic expression systems. In someembodiments, the expression system is a mammalian cell expression, suchas a CHO cell expression system. Many such systems are widely availablefrom commercial suppliers. In embodiments in which an antibody comprisesboth a V_(H) and V_(L) region, the V_(H) and V_(L) regions may beexpressed using a single vector, e.g., in a discistronic expressionunit, or under the control of different promoters. In other embodiments,the V_(H) and V_(L) region may be expressed using separate vectors. AV_(H) or V_(L) region as described herein may optionally comprise amethionine at the N-terminus.

An antibody of the invention may be produced in any number of formats,including as a Fab, a Fab′, a F(ab′)₂, a scFv, or a dAB. An antibody ofthe invention can also include a human constant region. The constantregion of the light chain may be a human kappa or lambda constantregion. The heavy chain constant region is often a gamma chain constantregion, for example, a gamma-1, gamma-2, gamma-3, or gamma-4 constantregion. In other embodiments, the antibody may be an IgA.

In some embodiments of the invention, the antibody V_(L) region, e.g.,VK#1, VK#2, VK#3, or VK#4 of FIG. 1, is combined with a human kappaconstant region (e.g., SEQ ID NO:10) to form the complete light-chain.

In some embodiments of the invention, the V_(H) region is combined ahuman gamma-1 constant region. Any suitable gamma-1 f allotype can bechosen, such as the f-allotype. Thus, in some embodiments, the antibodyis an IgG having an f-allotype constant region, e.g., SEQ ID NO:11, thathas a V_(H) selected from VH#1, VH#2, VH#3, VH#4, or VH#5 (FIG. 1). Insome embodiments, the antibody has a V_(L) selected from VK#1, VK#2,VK#3, or VK#4 (FIG. 1.) In particular embodiments, the antibody has akappa constant region as set forth in SEQ ID NO:10, and a heavy chainconstant region as set forth in SEQ ID NO:11, where the heavy and lightchain variable regions comprise one of the following combinations fromthe sequences set forth in FIG. 1: a) VH#2, VK#3; b) VH#1, VK#3; c)VH#3, VK#1; d) VH#3, VL#3; e) VH#4, VK#4; f) VH#4, VK#2; g) VH#5, VK#1;h) VH#5, VK#2; i) VH#3, VK#4; or j) VH#3, VL#3).

In some embodiments, e.g., where the antibody is a fragment, theantibody can be conjugated to another molecule, e.g., polyethyleneglycol (PEGylation) or serum albumin, to provide an extended half-lifein vivo. Examples of PEGylation of antibody fragments are provided inKnight et al. Platelets 15:409, 2004 (for abciximab); Pedley et al., Br.J. Cancer 70:1126, 1994 (for an anti-CEA antibody); Chapman et al.,Nature Biotech. 17:780, 1999; and Humphreys, et al., Protein Eng. Des.20: 227, 2007).

In some embodiments, the antibodies of the invention are in the form ofa Fab′ fragment. A full-length light chain is generated by fusion of aV_(L)-region to human kappa or lambda constant region. Either constantregion may be used for any light chain; however, in typical embodiments,a kappa constant region is used in combination with a Vkappa variableregion and a lambda constant region is used with a Vlambda variableregion.

The heavy chain of the Fab′ is a Fd′ fragment generated by fusion of aV_(H)-region of the invention to human heavy chain constant regionsequences, the first constant (CH1) domain and hinge region. The heavychain constant region sequences can be from any of the immunoglobulinclasses, but is often from an IgG, and may be from an IgG1, IgG2, IgG3or IgG4. The Fab′ antibodies of the invention may also be hybridsequences, e.g., a hinge sequence may be from one immunoglobulinsub-class and the CH1 domain may be from a different sub-class.

V. Administration of Anti-GM-CSF Antibodies for the Treatment ofDiseases in which GM-CSF is a Target.

The invention also provides methods of treating a patient that has adisease involving GM-CSF in which it is desirable to inhibit GM-CSFactivity, i.e., in which GM-CSF is a therapeutic target. In someembodiments, such a patient has a chronic inflammatory disease, e.g.,arthritis, e.g., rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, juvenile idiopathic arthritis, and other inflammatorydiseases of the joint; inflammatory bowel diseases, e.g., ulcerativecolitis, Crohn's disease, Barrett's syndrome, ileitis, enteritis, andgluten-sensitive enteropathy; inflammatory disorders of the respiratorysystem, such as asthma, adult respiratory distress syndrome, allergicrhinitis, silicosis, chronic obstructive pulmonary disease,hypersensitivity lung diseases, bronchiectasis; inflammatory diseases ofthe skin, including psoriasis, scleroderma, and inflammatory dermatosessuch as eczema, atopic dermatitis, urticaria, and pruritis; disordersinvolving inflammation of the central and peripheral nervous system,including multiple sclerosis, idiopathic demyelinating polyneuropathy,Guillain-Barre syndrome, chronic inflammatory demyelinatingpolyneuropathy, and neurodegenerative diseases such as Alzheimer'sdisease. Various other inflammatory diseases can be treated using themethods of the invention. These include systemic lupus erythematosis,immune-mediated renal disease, e.g., glomerulonephritis, andspondyloarthropathies; and diseases with an undesirable chronicinflammatory component such as systemic sclerosis, idiopathicinflammatory myopathies, Sjogren's syndrome, vasculitis, sarcoidosis,thyroiditis, gout, otitis, conjunctivitis, sinusitis, sarcoidosis,Behcet's syndrome, hepatobiliary diseases such as hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis.In some embodiments, the patient has inflammation following injury tothe cardiovascular system. Various other inflammatory diseases includetuberculosis and chronic cholecystitis. Additional chronic inflammatorydiseases are described, e.g., in Harrison's Principles of InternalMedicine, 12th Edition, Wilson, et al., eds., McGraw-Hill, Inc.). Insome embodiments, a patient treated with an antibody has a cancer inwhich GM-CSF contributes to tumor or cancer cell growth, e.g., acutemyeloid leukemia. In some embodiments, a patient treated with anantibody of the invention has, or is at risk of heart failure, e.g., dueto ischemic injury to the cardiovascular system such as ischemic heartdisease, stroke, and atherosclerosis. In some embodiments, a patienttreated with an antibody of the invention has asthma. In someembodiments, a patient treated with an antibody of the invention hasAlzheimer's disease. In some embodiments, a patient treated with anantibody of the invention has osteopenia, e.g., osteoporosis. In someembodiments, a patient treated with an antibody of the invention hasthrombocytopenia purpura. In some embodiments, the patient has Type I orType II diabetes. In some embodiments, a patient may have more than onedisease in which GM-CSF is a therapeutic target, e.g., a patient mayhave rheumatoid arthritis and heart failure, or osteoporosis andrheumatoid arthritis, etc.

The methods of the invention comprise administering an anti-GM-CSFantibody as a pharmaceutical composition to a patient in atherapeutically effective amount using a dosing regimen suitable fortreatment of the disease. The composition can be formulated for use in avariety of drug delivery systems. One or more physiologically acceptableexcipients or carriers can also be included in the compositions forproper formulation. Suitable formulations for use in the presentinvention are found in Remington: The Science and Practice of Pharmacy,21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins, 2005.

The anti-GM-CSF antibody is provided in a solution suitable forinjection into the patient such as a sterile isotonic aqueous solutionfor injection. The antibody is dissolved or suspended at a suitableconcentration in an acceptable carrier. In some embodiments the carrieris aqueous, e.g., water, saline, phosphate buffered saline, and thelike. The compositions may contain auxiliary pharmaceutical substancesas required to approximate physiological conditions, such as pHadjusting and buffering agents, tonicity adjusting agents, and the like.

The pharmaceutical compositions of the invention are administered to apatient, e.g., a patient that has osteopenia, asthma, rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, juvenileidiopathic arthritic, polymyositis, systemic lupus erythrematosus, heartfailure, cardiac damage, e.g., following a heart attack,thrombocytopenia purpura, or diabetes, in an amount sufficient to cureor at least partially arrest the disease or symptoms of the disease andits complications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” A therapeutically effective dose isdetermined by monitoring a patient's response to therapy. Typicalbenchmarks indicative of a therapeutically effective dose includeamelioration of symptoms of the disease in the patient. Amountseffective for this use will depend upon the severity of the disease andthe general state of the patient's health, including other factors suchas age, weight, gender, administration route, etc. Single or multipleadministrations of the antibody may be administered depending on thedosage and frequency as required and tolerated by the patient. In anyevent, the methods provide a sufficient quantity of anti-GM-CSF antibodyto effectively treat the patient.

The antibody may be administered alone, or in combination with othertherapies to treat the disease of interest.

The antibody can be administered by injection or infusion through anysuitable route including but not limited to intravenous, sub-cutaneous,intramuscular or intraperitoneal routes. In some embodiments, theantibody may be administered by insufflation. In an exemplaryembodiment, the antibody may be stored at 10 mg/ml in sterile isotonicaqueous saline solution for injection at 4° C. and is diluted in either100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the patient. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered byintravenous infusion over a period of between 15 minutes and 2 hours. Instill other embodiments, the administration procedure is viasub-cutaneous bolus injection.

The dose of antibody is chosen in order to provide effective therapy forthe patient and is in the range of less than 0.1 mg/kg body weight toabout 25 mg/kg body weight or in the range 1 mg-2 g per patient.Preferably the dose is in the range 1-10 mg/kg or approximately 50mg-1000 mg/patient. The dose may be repeated at an appropriate frequencywhich may be in the range once per day to once every three months,depending on the pharmacokinetics of the antibody (e.g., half-life ofthe antibody in the circulation) and the pharmacodynamic response (e.g.,the duration of the therapeutic effect of the antibody). In someembodiments, the in vivo half-life of between about 7 and about 25 daysand antibody dosing is repeated between once per week and once every 3months. In other embodiments, the antibody is administered approximatelyonce per month.

A V_(H) region and/or V_(L) region of the invention may also be used fordiagnostic purposes. For example, the V_(H) and/or V_(L) region may beused for clinical analysis, such as detection of GM-CSF levels in apatient. A V_(H) or V_(L) region of the invention may also be used,e.g., to produce anti-Id antibodies.

EXAMPLES Example 1 Identification of Engineered Human Anti-GM-CSF VRegions

Humaneering was performed as described in US patent applicationpublication no. 20050255552. Epitope-focused libraries were constructedfrom human V-segment library sequences linked to a CDR3-FR4 regioncontaining BSD sequences in CDRH3 and CDRL3 together with humangerm-line J-segment sequences. For the heavy chain, human germ-line JH4sequence was used and for the light chain, human germ-line JK4 sequencewas used.

Full-length Humaneered V-regions from a Vh1-restricted library wereselected that supported binding to recombinant human GM-CSF. The“full-length” V-kappa library was used as a base for construction of“cassette” libraries as described in US patent application publicationno. 20060134098, in which only part of the murine c19/2 V-segment wasinitially replaced by a library of human sequences. Two types ofcassettes were constructed. Cassettes for the V-kappa chains were madeby bridge PCR with overlapping common sequences within the framework 2region. In this way “front-end” and “middle” human cassette librarieswere constructed for the human V-kappa III isotype. Human V-kappa IIIcassettes which supported binding to GM-CSF were identified bycolony-lift binding assay and ranked according to affinity in ELISA. TheV-kappa human “front-end” and “middle” cassettes were fused together bybridge PCR to reconstruct a fully human V-kappa region that supportedGM-CSF binding activity. The Humaneered Fabs thus consist of HumaneeredV-heavy and V-kappa regions that support binding to human GM-CSF.

Binding activity was determined by surface plasmon resonance (spr)analysis. Biotinylated GM-CSF was captured on a streptavidin-coated CM5biosensor chip. Humaneered Fab fragments expressed from E. Coli werediluted to a starting concentration of 30 nM in 10 mM HEPES, 150 mMNaCl, 0.1 mg/ml BSA and 0.005% P20 at pH 7.4. Each Fab was diluted 4times using a 3-fold dilution series and each concentration was testedtwice at 37° C. to determine the binding kinetics with the differentdensity antigen surfaces. The data from all three surfaces were fitglobally to extract the dissociation constants.

Exemplary humaneered anti-GM-CSF V regions are shown in FIG. 1.

Example 2 Evaluation of a Humaneered GM-CSF Antibody

This example evaluates the binding activity and biological potency of ahumaneered anti-GM-CSF antibody in a cell-based assay in comparison to achimeric IgG1k antibody (Ab2) having variable regions from the mouseantibody LMM102 (Nice et al., Growth Factors 3:159, 1990). Ab1 is ahumaneered IgG1k antibody against GM-CSF having identical constantregions to Ab2.

Surface Plasmon Resonance Analysis of Binding of Human GM-CSF to Ab1 andAb2

Surface Plasmon resonance analysis was used to compare binding kineticsand monovalent affinities for the interaction of Ab1 and Ab2 withglycosylated human GM-CSF using a Biacore 3000 instrument. Ab1 or Ab2was captured onto the Biacore chip surface using polyclonal anti-humanF(ab′)2. Glycosylated recombinant human GM-CSF expressed from human 293cells was used as the analyte. Kinetic constants were determined in 2independent experiments (see FIG. 2 and Table 1). The results show thatGM-CSF bound to Ab2 and Ab1 with comparable monovalent affinity in thisexperiment. However, Ab1 had a two-fold slower “on-rate” than Ab2, butan “off-rate” that was approximately three-fold slower.

TABLE 1 Kinetic constants at 37° C. determined from the surface plasmonresonance analysis in FIG. 2; association constant (k_(a)), dissociationconstant (k_(d)) and calculated affinity (KD) are shown. k_(a) (M⁻¹s⁻¹)k_(d) (s⁻¹) KD (pM) Ab2 7.20 × 10⁵  2.2 × 10⁻⁵ 30.5 Ab1 2.86 × 10⁵ 7.20× 10⁻⁶ 25.1

GM-CSF is naturally glycosylated at both N-linked and O-linkedglycosylation sites although glycosylation is not required forbiological activity. In order to determine whether GM-CSF glycosylationaffects the binding of Ab1 or Ab2, the antibodies were compared in anELISA using recombinant GM-CSF from two different sources; GM-CSFexpressed in E. coli (non-glycosylated) and GM-CSF expressed from human293 cells (glycosylated). The results in FIG. 3 and Table 2 showed thatboth antibodies bound glycosylated and non-glycosylated GM-CSF withequivalent activities. The two antibodies also demonstrated comparableEC₅₀ values in this assay.

TABLE 2 Summary of EC₅₀ for binding of Ab2 and Ab1 to human GM-CSF fromtwo different sources determined by ELISA. Non-glycosylatedNon-glycosylated Glycosylated (exp 1) (exp 2) (exp 1) Ab2 400 pM 433 pM387 pM Ab1 373 pM 440 pM 413 pM Binding to recombinant GM-CSF from human293 cells (glycosylated) or from E. coli (non-glycosylated) wasdetermined from two independent experiments. Experiment 1 is shown inFIG. 5.

Ab1 is a humaneered antibody that was derived from the mouse variableregions present in Ab2. Ab1 was tested for overlapping epitopespecificity (Ab2) by competition ELISA.

Biotinylated Ab2 was prepared using known techniques. Biotinylation didnot affect binding of Ab2 to GM-CSF as determined by ELISA. In theassay, Ab2 or Ab1 was added in varying concentrations with a fixedamount of biotinylated Ab2. Detection of biotinylated Ab2 was assayed inthe presence of unlabeled Ab or Ab1 competitor (FIG. 4). Both Ab1 andAb2 competed with biotinylated Ab2 for binding to GM-CSF, thusindicating binding to the same epitope. Ab1 competed more effectivelyfor binding to GM-CSF than Ab2, consistent with the slower dissociationkinetics for Ab1 when compared with Ab2 by surface plasmon resonanceanalysis.

Neutralization of GM-CSF Activity by Ab1 and Ab2

A cell based assay for neutralization of GM-CSF activity was employed toevaluate biological potency. The assay measures IL-8 secretion from U937cells induced with GM-CSF. IL-8 secreted into the culture supernatant isdetermined by ELISA after 16 hours induction with 0.5 ng/ml E.coli-derived GM-CSF.

A comparison of the neutralizing activity of Ab1 and Ab2 in this assayis shown in a representative assay in FIG. 5. In three independentexperiments, Ab1 inhibited GM-CSF activity more effectively than Ab2when comparing IC₅₀ (Table 3).

TABLE 3 Comparison of IC50 for inhibition of GM-CSF induced IL-8expression. Experiment Ab2 (ng/ml) Ab2 (nM) Ab1 (ng/ml) Ab1 (nM) A 3632.4 31.3 0.21 B 514 3.4 92.5 0.62 C 343 2.2 20.7 0.14 Mean 407 2.7 48.20.32 Data from three independent experiments shown in FIG. 5 and meanIC₅₀ are expressed in ng/ml and nM.SummaryThe humaneered Ab1 bound to GM-CSF with a calculated equilibrium bindingconstant (KD) of 25 pM. Ab2 bound to GM-CSF with a KD of 30.5 pM. Ab2showed a two-fold higher association constant (k_(a)) than Ab1 forGM-CSF while Ab1 showed three-fold slower dissociation kinetics (k_(d))than Ab2. Ab2 and Ab1 showed similar binding activity for glycosylatedand non-glycosylated GM-CSF in an antigen-binding ELISA. A competitionELISA confirmed that both antibodies competed for the same epitope; Ab1showed higher competitive binding activity than Ab2. In addition, Ab1showed higher GM-CSF neutralization activity than Ab2 in aGM-CSF-induced IL-8 induction assay.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, accession numbers, patents, and patent applicationscited in this specification are herein incorporated by reference as ifeach was specifically and individually indicated to be incorporated byreference.

Exemplary V_(H )region sequences of anti-GM-CSFantibodies of the invention: SEQ ID NO: 1 (VH#1, FIG. 1)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRRD RFPYYFDYWGQGTLVTVSSSEQ ID NO: 2 (VH#2, FIG. 1)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRD RFPYYFDYWGQGTLVTVSSSEQ ID NO: 3 (VH#3, FIG. 1)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRQ RFPYYFDYWGQGTLVTVSSSEQ ID NO: 4 (VH#4, FIG. 1)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCVRRQ RFPYYFDYWGQGTLVTVSSSEQ ID NO: 5 (VH#5, FIG. 1)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKEQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRRQ RFPYYFDYWGQGTLVTVSSExemplary V_(L )region sequences of anti-GM-CSFantibodies of the invention: SEQ ID NO: 6 (VK#1, FIG. 1)EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGG GTKVEIK SEQ ID NO: 7(VK#2, FIG. 1) EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGG GTKVEIK SEQ ID NO: 8(VK#3, FIG. 1) EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGG GTKVEIK SEQ ID NO: 9(VK#4, FIG. 1) EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGG GTKVEIK SEQ ID NO: 10Exemplary kappa constant regionRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC SEQ ID NO: 11Exemplary heavy chain constant region, f-allotype:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1. An isolated anti-GM-CSF antibody, comprising: a V_(H) that has a CDR1having a sequence NYYIH (SEQ ID NO:25), a CDR2 having a sequenceWINAGNGNTKYSQKFQG (SEQ ID NO:27), and a CDR3 having a sequenceRQRFPYYFDY (SEQ ID NO:16); and a V_(L) that has a CDR1 having a sequenceRASQSVGTNVA (SEQ ID NO:31) or RASQSIGSNLA (SEQ ID NO:32), a CDR2 havinga sequence STSSRAT (SEQ ID NO:33), and a CDR3 having a sequenceQQFNKSPLT (SEQ ID NO:18) or QQFNRSPLT (SEQ ID NO:28).
 2. The antibody ofclaim 1, wherein the J segment comprises YFDYWGQGTLVTVSS (SEQ ID NO:14).3. The antibody of claim 1, wherein the V_(H) region comprises aV-segment sequence from a V_(H) region VH#3 (SEQ ID NO:3), VH#4 (SEQ IDNO:4), or VH#5 (SEQ ID NO:5).
 4. The antibody of claim 1, wherein theV_(H) has the sequence of VH#3 (SEQ ID NO:3), VH#4 (SEQ ID NO:4), orVH#5 (SEQ ID NO:5).
 5. The antibody of claim 1, wherein the antibodycomprises a human germline JK4 region.
 6. The antibody of claim 1,wherein the V_(L) region CDR3 comprises QQFNKSPLT (SEQ ID NO:18).
 7. Theantibody of claim 6, wherein the V_(L) region CDR1 has the sequenceRASQSIGSNLA (SEQ ID NO:32).
 8. The antibody of claim 1, wherein theV_(L) region CDR3 comprises QQFNRSPLT (SEQ ID NO:28).
 9. The antibody ofclaim 8, wherein the V_(L) region CDR1 has the sequence RASQSVGTNVA (SEQID NO:31).
 10. The antibody of claim 1, wherein the V_(L) regioncomprises a V segment sequence from a V_(L) region VK#1 (SEQ ID NO:6),VK#2 (SEQ ID NO:7), VK#3 (SEQ ID NO:8), or VK#4 (SEQ ID NO:9).
 11. Theantibody of claim 1, wherein the V_(L) region has the sequence of VK#1(SEQ ID NO:6), VK#2 (SEQ ID NO:7), VK#3 (SEQ ID NO:8), or VK#4 (SEQ IDNO:9).
 12. The antibody of claim 1, wherein the antibody is an IgG. 13.The antibody of claim 12, wherein the antibody comprises a heavy chainconstant region having the amino acid sequence set forth in SEQ IDNO:11.
 14. The antibody of claim 1, wherein the antibody comprises akappa light chain constant region having the amino acid sequence setforth in SEQ ID NO:10.
 15. The antibody of claim 1, wherein the V_(H)region or the V_(L) region, or both the V_(H) and V_(L) region aminoacid sequences comprise a methionine at the N-terminus.
 16. The antibodyof claim 1, wherein the antibody comprises a V_(H) region having asequence of VH#3 (SEQ ID NO:3), VH#4 (SEQ ID NO:4), or VH#5 (SEQ IDNO:5) and a V_(L) region having a sequence of VK#1 (SEQ ID NO:6), VK#2(SEQ ID NO:7), VK#3 (SEQ ID NO:8), or VK#4 (SEQ ID NO:9).
 17. Theantibody of claim 16, wherein the antibody is an IgG.
 18. The antibodyof claim 17, wherein the antibody comprises a heavy chain constantregion having the amino acid sequence set forth in SEQ ID NO:11.
 19. Theantibody of claim 16, wherein the antibody comprises a kappa light chainconstant region having the amino acid sequence set forth in SEQ IDNO:10.
 20. The antibody of claim 1, wherein the antibody has: a heavychain and a light chain variable region combination selected from thegroup consisting of: a) VH#4 (SEQ ID NO:4), VK#1 (SEQ ID NO:6); b) VH#4(SEQ ID NO:4), VK#2 (SEQ ID NO:7; c) VH#4 (SEQ ID NO:4), VK#3 (SEQ IDNO:8) d) VH#4 (SEQ ID NO:4), VK#4 (SEQ ID NO:9); e) VH#5 (SEQ ID NO:5),VK#1 (SEQ ID NO:6); f) VH#5 (SEQ ID NO:5), VK#2 (SEQ ID NO:7; g) VH#5(SEQ ID NO:5), VK#3 (SEQ ID NO:8); and d) VH#5 (SEQ ID NO:5), VK#4 (SEQID NO:9).
 21. The antibody of claim 20, wherein the heavy chain variableregion is VH#4 (SEQ ID NO:4) and the light chain variable region is VK#1(SEQ ID NO:6).
 22. The antibody of claim 20, wherein the heavy chainvariable region is VH#4 (SEQ ID NO:4) and the light chain variableregion is VK#2 (SEQ ID NO:7).
 23. The antibody of claim 20, wherein theheavy chain variable region is VH#4 (SEQ ID NO:4) and the light chainvariable region is VK#3 (SEQ ID NO:8).
 24. The antibody of claim 20,wherein the heavy chain variable region is VH#4 (SEQ ID NO:4) and thelight chain variable region is VK#4 (SEQ ID NO:9).
 25. The antibody ofclaim 20, wherein the heavy chain variable region is VH#5 (SEQ ID NO:5)and the light chain variable region is VK#1 (SEQ ID NO:6).
 26. Theantibody of claim 20, wherein the heavy chain variable region is VH#5(SEQ ID NO:5) and the light chain variable region is VK#2 (SEQ ID NO:7).27. The antibody of claim 20, wherein the heavy chain variable region isVH#5 (SEQ ID NO:5) and the light chain variable region is VK#3 (SEQ IDNO:8).
 28. The antibody of claim 20, wherein the heavy chain variableregion is VH#5 (SEQ ID NO:5) and the light chain variable region is VK#4(SEQ ID NO:9).
 29. The antibody of claim 20, wherein the antibodycomprises a heavy chain constant region having the amino acid sequenceset forth in SEQ ID NO:11.
 30. The antibody of claim 29, wherein theantibody comprises a kappa light chain constant region having the aminoacid sequence set forth in SEQ ID NO:10.
 31. An isolated anti-GM-CSFantibody comprising a V_(H) region and a V_(L) region, wherein the V_(H)region has the amino acid sequence set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
 32. The isolatedantibody of claim 31 wherein the antibody comprises the V_(H) region setforth in SEQ ID NO:5.
 33. The isolated antibody of claim 31 wherein theantibody comprises the V_(H) region set forth in SEQ ID NO:4.
 34. Theisolated antibody of claim 31 wherein the antibody comprises the V_(H)region set forth in SEQ ID NO:3.
 35. The isolated antibody of claim 31wherein the antibody comprises the V_(H) region set forth in SEQ IDNO:2.
 36. The isolated antibody of claim 31 wherein the antibodycomprises the V_(H) region set forth in SEQ ID NO:1.